Monoclonal antibodies were used to study antigenic differences among strains of ADV and to characterize viral proteins in vitro and in vivo. Highly virulent Utah I ADV was clearly delineated from the tissue culture-adapted avirulent ADV-G strain. This specificity could be demonstrated by indirect immunofluorescence (IFA) against infected cultures of Crandell feline kidney cells or against tissues of Utah I ADV-infected mink. Immunoprecipitation analyses utilizing various mAbs identified specific antigenic determinants. When immunoprecipitation- defined reactivities were correlated with IFA tissue and in vitro patterns of reactivity it was apparent that the virus-associated antigenic determinants recognized in vivo were proteolytic products of viral structural proteins. Thus, intact structural ADV proteins were not identified in vivo. However, structural proteins were detected in vitro when ADV-G or Utah I ADV-infected CRFK cells were analyzed.